A third gasotransmitter, joining NO and CO, is hydrogen sulfide (H2S) that is produced physiologically from L-cysteine catabolized by cystathionine 3-lyase (CSE) and cystathionine 2-synthase (CBS). CSE is the predominant isoform in the vasculature and CBS in whole brain. Reports are emerging of roles of hydrogen sulfide (H2S) in neuronal function in the brain, but no published data exist on H2S and cerebrovascular function. The research proposed is designed to pursue the unifying hypothesis that H2S synthesized by endothelial CSE is an integral component of neonatal cerebral circulatory regulation. To test this hypothesis, four specific aims will be addressed in newborn pigs: 1. Determine the functional significance of H2S in regulation of cerebral arteriolar resistance, 2. Localize and characterize H2S production in the neonatal cerebral vasculature, 3. Investigate, in vivo, the contribution of endothelium to H2S-dependent cerebrovascular responses, and 4. Investigate mechanisms of H2S-induced modifications of cerebral arteriolar tone. To accomplish these aims, techniques will be used that allow investigation of intact newborn cerebral microcirculation in vivo, pressurized isolated cerebral arterioles, freshly isolated cerebral microvessels and smooth muscle cells, and cerebral microvascular endothelial cells in primary culture. Such research is unique by studying intact cerebral circulation and investigating, at the cellular and molecular levels, the mechanisms responsible for controlling the production of the mediator, H2S, and the mechanisms by which H2S can affect vascular tone. Cranial windows allow observation of cerebral circulation, collection of cortical periarachnoid fluid, and topical application of agonists, precursors and inhibitors. Cellular distribution of CSE and CBS and cellular mechanisms for controlling the enzyme activity will be examined. As the indicator of the enzyme activity, we propose a novel analytical method of H2S detection, identification, and quantification by gas chromatography-mass spectrometry. The cellular mechanisms by which H2S may modify vascular tone will be studied at the level of second messengers, ion channels, and membrane potential. Disorders of the cerebral circulation in the newborn period are major causes of morbidity and mortality and can result in lifelong disabilities in survivors. Control of cerebrovascular circulation is easily impaired by pathological conditions. Better understanding of mechanisms of cerebromicrovascular humoral communication in newborns is needed badly. PUBLIC HEALTH RELEVANCE: The single most prominent cause of mortality and morbidity in newborns is hypoxic-ischemic brain injury, which often leads to lifelong disability. Successes in developing approaches to avert and treat perinatal hypoxia- ischemic brain damage have been limited by insufficient understanding of the mechanisms that control perinatal cerebral circulation. The gasotransmitter, H2S, will certainly prove to be one of these major mechanisms of which greater understanding is urgently needed.